Genetic determinism of spontaneous masculinisation in XX female rainbow trout: new insights using medium throughput genotyping and whole-genome sequencing.

Rainbow trout has a male heterogametic (XY) sex determination system controlled by a major sex-determining gene, sdY. Unexpectedly, a few phenotypically masculinised fish are regularly observed in all-female farmed trout stocks. To better understand the genetic determinism underlying spontaneous maleness in XX-rainbow trout, we recorded the phenotypic sex of 20,210 XX-rainbow trout from a French farm population at 10 and 15 months post-hatching. The overall masculinisation rate was 1.45%. We performed two genome-wide association studies (GWAS) on a subsample of 1139 individuals classified as females, intersex or males using either medium-throughput genotyping (31,811 SNPs) or whole-genome sequencing (WGS, 8.7 million SNPs). The genomic heritability of maleness ranged between 0.48 and 0.62 depending on the method and the number of SNPs used for the estimation. At the 31K SNPs level, we detected four QTL on three chromosomes (Omy1, Omy12 and Omy20). Using WGS information, we narrowed down the positions of the two QTL detected on Omy1 to 96 kb and 347 kb respectively, with the second QTL explaining up to 14% of the total genetic variance of maleness. Within this QTL, we detected three putative candidate genes, fgfa8, cyp17a1 and an uncharacterised protein (LOC110527930), which might be involved in spontaneous maleness of XX-female rainbow trout.

Correction to: Quantitative trait loci for resistance to Flavobacterium psychrophilum in rainbow trout: effect of the mode of infection and evidence of epistatic interactions.

After publication of this work [1], we noted that there was an error in Table 3 Line 4.

Quantitative trait loci for resistance to Flavobacterium psychrophilum in rainbow trout: effect of the mode of infection and evidence of epistatic interactions.

BACKGROUND: Bacterial cold-water disease, which is caused by Flavobacterium psychrophilum, is one of the major diseases that affect rainbow trout (Oncorhynchus mykiss) and a primary concern for trout farming. Better knowledge of the genetic basis of resistance to F. psychrophilum would help to implement this trait in selection schemes and to investigate the immune mechanisms associated with resistance. Various studies have revealed that skin and mucus may contribute to response to infection. However, previous quantitative trait loci (QTL) studies were conducted by using injection as the route of infection. Immersion challenge, which is assumed to mimic natural infection by F. psychrophilum more closely, may reveal different defence mechanisms. RESULTS: Two isogenic lines of rainbow trout with contrasting susceptibilities to F. psychrophilum were crossed to produce doubled haploid F2 progeny. Fish were infected with F. psychrophilum either by intramuscular injection (115 individuals) or by immersion (195 individuals), and genotyped for 9654 markers using RAD-sequencing. Fifteen QTL associated with resistance traits were detected and only three QTL were common between the injection and immersion. Using a model that accounted for epistatic interactions between QTL, two main types of interactions were revealed. A "compensation-like" effect was detected between several pairs of QTL for the two modes of infection. An "enhancing-like" interaction effect was detected between four pairs of QTL. Integration of the QTL results with results of a previous transcriptomic analysis of response to F. psychrophilum infection resulted in a list of potential candidate immune genes that belong to four relevant functional categories (bacterial sensors, effectors of antibacterial immunity, inflammatory factors and interferon-stimulated genes). CONCLUSIONS: These results provide new insights into the genetic determinism of rainbow trout resistance to F. psychrophilum and confirm that some QTL with large effects are involved in this trait. For the first time, the role of epistatic interactions between resistance-associated QTL was evidenced. We found that the infection protocol used had an effect on the modulation of defence mechanisms and also identified relevant immune functional candidate genes.

Genetic and transcriptomic analyses provide new insights on the early antiviral response to VHSV in resistant and susceptible rainbow trout.

BACKGROUND: The viral hemorrhagic septicemia virus (VHSV) is a major threat for salmonid farming and for wild fish populations worldwide. Previous studies have highlighted the importance of innate factors regulated by a major quantitative trait locus (QTL) for the natural resistance to waterborne VHSV infection in rainbow trout. The aim of this study was to analyze the early transcriptomic response to VHSV inoculation in cell lines derived from previously described resistant and susceptible homozygous isogenic lines of rainbow trout to obtain insights into the molecular mechanisms responsible for the resistance to the viral infection. RESULTS: We first confirmed the presence of the major QTL in a backcross involving a highly resistant fish isogenic line (B57) and a highly susceptible one (A22), and were able to define the confidence interval of the QTL and to identify its precise position. We extended the definition of the QTL since it controls not only resistance to waterborne infection but also the kinetics of mortality after intra-peritoneal injection. Deep sequencing of the transcriptome of B57 and A22 derived cell lines exposed to inactivated VHSV showed a stronger response to virus inoculation in the resistant background. In line with our previous observations, an early and strong induction of interferon and interferon-stimulated genes was correlated with the resistance to VHSV, highlighting the major role of innate immune factors in natural trout resistance to the virus. Interestingly, major factors of the antiviral innate immunity were much more expressed in naive B57 cells compared to naive A22 cells, which likely contributes to the ability of B57 to mount a fast antiviral response after viral infection. These observations were further extended by the identification of several innate immune-related genes localized close to the QTL area on the rainbow trout genome. CONCLUSIONS: Taken together, our results improve our knowledge in virus-host interactions in vertebrates and provide novel insights in the molecular mechanisms explaining the resistance to VHSV in rainbow trout. Our data also provide a collection of potential markers for resistance and susceptibility of rainbow trout to VHSV infection.

A synthetic rainbow trout linkage map provides new insights into the salmonid whole genome duplication and the conservation of synteny among teleosts.

BACKGROUND: Rainbow trout is an economically important fish and a suitable experimental organism in many fields of biology including genome evolution, owing to the occurrence of a salmonid specific whole-genome duplication (4th WGD). Rainbow trout is among some of the most studied teleosts and has benefited from substantial efforts to develop genomic resources (e.g., linkage maps. Here, we first generated a synthetic map by merging segregation data files derived from three independent linkage maps. Then, we used it to evaluate genome conservation between rainbow trout and three teleost models, medaka, stickleback and zebrafish and to further investigate the extent of the 4th WGD in trout genome. RESULTS: The INRA linkage map was updated by adding 211 new markers. After standardization of marker names, consistency of marker assignment to linkage groups and marker orders was checked across the three different data sets and only loci showing consistent location over all or almost all of the data sets were kept. This resulted in a synthetic map consisting of 2226 markers and 29 linkage groups spanning over 3600 cM. Blastn searches against medaka, stickleback, and zebrafish genomic databases resulted in 778, 824 and 730 significant hits respectively while blastx searches yielded 505, 513 and 510 significant hits. Homology search results revealed that, for most rainbow trout chromosomes, large syntenic regions encompassing nearly whole chromosome arms have been conserved between rainbow trout and its closest models, medaka and stickleback. Large conserved syntenies were also found between the genomes of rainbow trout and the reconstructed teleost ancestor. These syntenies consolidated the known homeologous affinities between rainbow trout chromosomes due to the 4th WGD and suggested new ones. CONCLUSIONS: The synthetic map constructed herein further highlights the stability of the teleost genome over long evolutionary time scales. This map can be easily extended by incorporating new data sets and should help future rainbow trout whole genome sequence assembly. Finally, the persistence of large conserved syntenies across teleosts should facilitate the identification of candidate genes through comparative mapping, even if the occurrence of intra-chromosomal micro-rearrangement may hinder the accurate prediction their genomic location.

Detection of QTL with effects on osmoregulation capacities in the rainbow trout (Oncorhynchus mykiss).

BACKGROUND: There is increasing evidence that the ability to adapt to seawater in teleost fish is modulated by genetic factors. Most studies have involved the comparison of species or strains and little is known about the genetic architecture of the trait. To address this question, we searched for QTL affecting osmoregulation capacities after transfer to saline water in a nonmigratory captive-bred population of rainbow trout. RESULTS: A QTL design (5 full-sib families, about 200 F2 progeny each) was produced from a cross between F0 grand-parents previously selected during two generations for a high or a low cortisol response after a standardized confinement stress. When fish were about 18 months old (near 204 g body weight), individual progeny were submitted to two successive hyper-osmotic challenges (30 ppt salinity) 14 days apart. Plasma chloride and sodium concentrations were recorded 24 h after each transfer. After the second challenge, fish were sacrificed and a gill index (weight of total gill arches corrected for body weight) was recorded. The genome scan was performed with 196 microsatellites and 85 SNP markers. Unitrait and multiple-trait QTL analyses were carried out on the whole dataset (5 families) through interval mapping methods with the QTLMap software. For post-challenge plasma ion concentrations, significant QTL (P < 0.05) were found on six different linkage groups and highly suggestive ones (P < 0.10) on two additional linkage groups. Most QTL affected concentrations of both chloride and sodium during both challenges, but some were specific to either chloride (2 QTL) or sodium (1 QTL) concentrations. Six QTL (4 significant, 2 suggestive) affecting gill index were discovered. Two were specific to the trait, while the others were also identified as QTL for post-challenge ion concentrations. Altogether, allelic effects were consistent for QTL affecting chloride and sodium concentrations but inconsistent for QTL affecting ion concentrations and gill morphology. There was no systematic lineage effect (grand-parental origin of QTL alleles) on the recorded traits. CONCLUSIONS: For the first time, genomic loci associated with effects on major physiological components of osmotic adaptation to seawater in a nonmigratory fish were revealed. The results pave the way for further deciphering of the complex regulatory mechanisms underlying seawater adaptation and genes involved in osmoregulatory physiology in rainbow trout and other euryhaline fishes.

A first generation integrated map of the rainbow trout genome.

BACKGROUND: Rainbow trout (Oncorhynchus mykiss) are the most-widely cultivated cold freshwater fish in the world and an important model species for many research areas. Coupling great interest in this species as a research model with the need for genetic improvement of aquaculture production efficiency traits justifies the continued development of genomics research resources. Many quantitative trait loci (QTL) have been identified for production and life-history traits in rainbow trout. An integrated physical and genetic map is needed to facilitate fine mapping of QTL and the selection of positional candidate genes for incorporation in marker-assisted selection (MAS) programs for improving rainbow trout aquaculture production. RESULTS: The first generation integrated map of the rainbow trout genome is composed of 238 BAC contigs anchored to chromosomes of the genetic map. It covers more than 10% of the genome across segments from all 29 chromosomes. Anchoring of 203 contigs to chromosomes of the National Center for Cool and Cold Water Aquaculture (NCCCWA) genetic map was achieved through mapping of 288 genetic markers derived from BAC end sequences (BES), screening of the BAC library with previously mapped markers and matching of SNPs with BES reads. In addition, 35 contigs were anchored to linkage groups of the INRA (French National Institute of Agricultural Research) genetic map through markers that were not informative for linkage analysis in the NCCCWA mapping panel. The ratio of physical to genetic linkage distances varied substantially among chromosomes and BAC contigs with an average of 3,033 Kb/cM. CONCLUSIONS: The integrated map described here provides a framework for a robust composite genome map for rainbow trout. This resource is needed for genomic analyses in this research model and economically important species and will facilitate comparative genome mapping with other salmonids and with model fish species. This resource will also facilitate efforts to assemble a whole-genome reference sequence for rainbow trout.

A type I and type II microsatellite linkage map of rainbow trout (Oncorhynchus mykiss) with presumptive coverage of all chromosome arms.

BACKGROUND: The development of large genomic resources has become a prerequisite to elucidate the wide-scale evolution of genomes and the molecular basis of complex traits. Linkage maps represent a first level of integration and utilization of such resources and the primary framework for molecular analyses of quantitative traits. Previously published linkage maps have already outlined the main peculiarities of the rainbow trout meiosis and a correspondance between linkage groups and chromosome arms has been recently established using fluorescent in situ hybridization. The number of chromosome arms which were covered by these maps remained unknown. RESULTS: We report an updated linkage map based on segregation analysis of more than nine hundred microsatellite markers in two doubled haploid gynogenetic lines. These markers segregated into 31 linkage groups spanning an approximate total map length of 2750 cM. Centromeres were mapped for all the linkage groups using meiogenetic lines. For each of the 31 linkage groups, the meta or acrocentric structure infered from centromere mapping was identical with those recently found with fluorescent in situ hybridization results. The present map is therefore assumed to cover the 52 chromosome arms which constitute the rainbow trout karyotype. Our data confirm the occurrence of a high interference level in this species. Homeologous regions were identified in eleven linkage groups, reflecting the tetraploid nature of the salmonid genome. The data supported the assumption that gene orders are conserved between duplicated groups and that each group is located on a single chromosome arm. Overall, a high congruence with already published rainbow trout linkage maps was found for both gene syntenies and orders. CONCLUSION: This new map is likely to cover the whole set of chromosome arms and should provide a useful framework to integrate existing or forthcoming rainbow trout linkage maps and other genomic resources. Since very large numbers of EST containing microsatellite sequences are available in databases, it becomes feasible to construct high-density linkage maps localizing known genes. This will facilitate comparative mapping and, eventually, identification of candidate genes in QTL studies.

A linkage map for brown trout (Salmo trutta): chromosome homeologies and comparative genome organization with other salmonid fish.

We report on the construction of a linkage map for brown trout (Salmo trutta) and its comparison with those of other tetraploid-derivative fish in the family Salmonidae, including Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), and Arctic char (Salvelinus alpinus). Overall, we identified 37 linkage groups (2n = 80) from the analysis of 288 microsatellite polymorphisms, 13 allozyme markers, and phenotypic sex in four backcross families. Additionally, we used gene-centromere analysis to approximate the position of the centromere for 20 linkage groups and thus relate linkage arrangements to the physical morphology of chromosomes. Sex-specific maps derived from multiple parents were estimated to cover 346.4 and 912.5 cM of the male and female genomes, respectively. As previously observed in other salmonids, recombination rates showed large sex differences (average female-to-male ratio was 6.4), with male crossovers generally localized toward the distal end of linkage groups. Putative homeologous regions inherited from the salmonid tetraploid ancestor were identified for 10 pairs of linkage groups, including five chromosomes showing evidence of residual tetrasomy (pseudolinkage). Map alignments with orthologous regions in Atlantic salmon, rainbow trout, and Arctic char also revealed extensive conservation of syntenic blocks across species, which was generally consistent with chromosome divergence through Robertsonian translocations.

Enhanced individual selection for selecting fast growing fish: the "PROSPER" method, with application on brown trout (Salmo trutta fario).

Growth rate is the main breeding goal of fish breeders, but individual selection has often shown poor responses in fish species. The PROSPER method was developed to overcome possible factors that may contribute to this low success, using (1) a variable base population and high number of breeders (Ne >100), (2) selection within groups with low non-genetic effects and (3) repeated growth challenges. Using calculations, we show that individual selection within groups, with appropriate management of maternal effects, can be superior to mass selection as soon as the maternal effect ratio exceeds 0.15, when heritability is 0.25. Practically, brown trout were selected on length at the age of one year with the PROSPER method. The genetic gain was evaluated against an unselected control line. After four generations, the mean response per generation in length at one year was 6.2% of the control mean, while the mean correlated response in weight was 21.5% of the control mean per generation. At the 4th generation, selected fish also appeared to be leaner than control fish when compared at the same size, and the response on weight was maximal (approximately 130% of the control mean) between 386 and 470 days post fertilisation. This high response is promising, however, the key points of the method have to be investigated in more detail.